PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis (TB), a disease that afflicts 2 billion people and results in 1.5 million deaths each year. The global burden of TB is enormous and growing, and an effective vaccine would provide the best long-term solution to this problem. However, such a vaccine still does not exist. Major barriers that have hindered the development of an effective TB vaccine include an incomplete understanding of which antigenic determinants confer protective immunity and a dearth of vaccination strategies capable of eliciting memory CD8+ and CD4+ T cell responses that can rapidly respond to and clear infection. The objective of this project is to design and evaluate an innovative nanoparticle (NP) vaccine engineered to elicit robust and durable pulmonary CD8+ and CD4+ Th1 memory T cell responses against Mtb antigens. NPs will be developed and optimized for dual-delivery of Mtb epitopes and 5' triphosphorylated RNA (5'ppp-RNA), an agonist of the cytosolic pattern recognition receptor (PRR) retinoic acid-inducible gene 1 (RIG-I) that has immense untapped potential as a vaccine adjuvant. Specifically, we propose to harness the unique delivery capabilities of NP vaccines to enhance pulmonary Th1 and CD8+ T cell responses to established and clinically relevant Mtb MHC class I- and class II-restricted epitopes as well as naturally processed class I-restricted epitopes recently discovered by our team. We have assembled a strong multidisciplinary team with expertise in drug delivery, vaccine design and development, RNA engineering, epitope discovery, and mouse models of Mtb infection. Therefore, we are ideally positioned to accomplish our objective through the following Specific Aims: 1) Develop a nanoparticle vaccine that elicits lung-resident CD8+ and Th1 memory T cell responses to Mtb subunit antigens; 2) Evaluate the immunogenicity and protective potential of naturally processed Mtb epitopes. In Aim 1, we will investigate the effect of intranasal NP immunization on the magnitude and kinetics of pulmonary antigen/adjuvant delivery and intracellular uptake, elucidate the immunostimulatory adjuvant effects of 5'ppp-RNA, and rigorously characterize pulmonary and systemic CD4+ and CD8+ T cell responses to Mtb antigens. In Aim 2, we will use NPs to deliver novel Mtb class I-restricted epitopes, both alone and in combination with an established immunodominant class II Mtb epitope, characterize local and systemic T cell responses, and assess the ability of vaccines to protect against pulmonary Mtb challenge. This project will result in a new vaccine technology for enhancing mucosal cellular immunity to subunit vaccines, will expand the armamentarium of vaccine adjuvants, and will provide a tool that empowers the discovery of immunogenic and protective Mtb epitopes. By combining epitope discovery efforts with rationally engineered delivery systems, successful completion of the proposed research will establish a new and potentially transformative paradigm for the rational design of T cell-targeted vaccines.